Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control

Katharine Y. Chen^1,2, Heungwon Park¹, Arvind Rasi Subramaniam^1,†

¹ Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA
² Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA

^† Corresponding author: rasi@fredhutch.org

Nucleic Acids Research 10.1093/nar/gkae285

Abstract

Decay of mRNAs can be triggered by ribosome slowdown at stretches of rare codons or positively charged amino acids. However, the full diversity of sequence motifs that trigger co-translational mRNA decay is poorly understood. To comprehensively identify sequence motifs that trigger mRNA decay, we use a massively parallel reporter assay to measure the effect of all possible combinations of codon pairs on mRNA levels in S. cerevisiae. In addition to known mRNA-destabilizing sequences, we identify several dipeptide repeats whose translation reduces mRNA levels. These include combinations of positively charged and bulky residues, as well as proline-glycine and proline-aspartic acid dipeptide repeats. Genetic deletion of the ribosome collision sensor Hel2 rescues the mRNA effects of these motifs, suggesting that they trigger ribosome slowdown and activate the ribosome-associated quality control (RQC) pathway. Deep mutational scanning of an mRNA-destabilizing dipeptide repeat reveals a complex relationship between the charge, bulkiness, and location of amino acid residues in conferring mRNA instability. Finally, we show that the mRNA effects of codon pairs are predictive of the effects of endogenous sequences. Our work highlights the complexity of sequence motifs driving co-translational mRNA decay in eukaryotes, and presents a high-throughput approach to dissect their requirements at the codon level.

• Abstract
• Instructions for running the code repo
• Data
  • Includes flow cytometry raw data that is also available at http://flowrepository.org/id/FR-FCM-Z6QH
• Code for designing endogenous fragments library
• Code for processing flow cytometry data and regenerating figure panels
• Code for linking barcodes and codon pair inserts in original 8× dicodon library
• Code for linking barcodes and codon pair inserts in frameshifted 8× dicodon library
• Code for linking barcodes and codon pair inserts in small-scale validation library
• Code for counting barcodes and regenerating figures:
  • codon pair library wild-type cells
  • codon pair library hel2Δ and syh1Δ cells
  • frameshifted codon pair library
  • small-scale validation library
  • codon pair library under glucose depletion
  • small codon pair library in RQC deletion strains
• Code for counting insert-UMI pairs in (FK)8 DMS library in wild-type and hel2∆ cells
• Code for counting insert-UMI pairs in (FK)8 DMS library in upf1∆_cells
• Code for linking barcodes and codon pair inserts in small 8× dicodon library in RQC deletion strains
• Code to regenerate all figure panels

Running the code

• To run this on a cluster with singularity containers, do:

module load singularity # for fred hutch cluster
conda activate snakemake # this is a minimal conda env that has snakemake-minimal and pandas for invoking snakefile
sh run_everything.sh

• The run_everything.sh file will:
  • Download FASTQ files from SRA
  • Run all linkage sequencing, barcode sequencing, and insert sequencing code
  • Run code to design of the endogenous fragments library
  • Run code to regenerate figure panels
    • This will also run the code to process flow cytometry data

Docker containers

• R
• python
• R and python

Code to generate figure panels from manuscript

Figure panels	Experiment	Script
1B, 1C, 1D, 1E, 1F	Barcode seq of wild-type cells with codon pair library	analysis/barcodeseq/wt_mrna_grna/scripts/plot_aggregate_effects.ipynb
2A, 2B, S1D	Barcode seq of wild-type cells with codon pair library	analysis/barcodeseq/wt_mrna_grna/scripts/plot_dipeptide_effects.ipynb
2C, 2D	Flow cytometry of wild-type cells with individual codon pair inserts	analysis/flow_cytometry/scripts/plot_figure2_flow.ipynb
3A	Barcode seq of wild-type cells with codon pair library computationally frameshifted	analysis/barcodeseq/wt_hel2_no_glucose_mrna_grna/scripts/plot_translation_effects.ipynb
3B, 3C, S4B	Barcode seq of wild-type cells with codon pair library during glucose depletion	analysis/barcodeseq/wt_hel2_no_glucose_mrna_grna/scripts/plot_translation_effects.ipynb
3E, 3F	Barcode seq of wild-type cells with -1 frameshifted codon pair library	analysis/barcodeseq/wt_hel2_no_glucose_mrna_grna/scripts/plot_translation_effects.ipynb
4C, 4D, 4E, S3C, S6A	Barcode seq of hel2∆ and syh1∆ cells with codon pair library	analysis/barcodeseq/hel2_syh1_mrna_grna/scripts/plot_hel2_syh1_dipeptide_effects.ipynb
5B, 5C, S5C	Deep mutational scan of (FK)8 in wild-type and hel2∆ cells	analysis/barcodeseq/wt_hel2_fk8_dms/scripts/plot_variant_effects.ipynb
6B, 6C, 6D	Barcode seq of wild-type and hel2∆ cells with endogenous fragments library	analysis/barcodeseq/endo_frag_mrna_grna/scripts/plot_endogenous_frags.ipynb
S1A	Barcode seq of wild-type cells with codon pair library	analysis/barcodeseq/wt_mrna_grna/scripts/plot_supp_alignment_stats.ipynb
S1B, S1C, S1E	Barcode seq of wild-type cells with codon pair library	analysis/barcodeseq/wt_mrna_grna/scripts/plot_supplemental_missing_data.ipynb
S2A, S2B	Barcode seq of wild-type with codon pair or mini-pool libraries	analysis/barcodeseq/wt_mrna_grna/scripts/plot_supp_alignment_stats.ipynb
S2C, S2D, S2E	Flow cytometry of wild-type and hel2∆ cells with individual codon pair inserts	analysis/barcodeseq/hel2_syh1_mrna_grna/scripts/plot_supp_aln_qc.ipynb
S3A, S3B	Summary of wild-type cells with codon pair library during all translation-related conditions	analysis/barcodeseq/wt_hel2_no_glucose_mrna_grna/scripts/plot_translation_effects.ipynb
S4A	Wild-type, hel2∆, syh1∆, hel2∆/syh1∆, cue2∆, and xrn1∆ cells with revision codon pair pool	analysis/barcodeseq/small_8xdicodon_rqcdel_mrna_grna/scripts/plot_dicodon_effects.ipynb
S5A, S5B	Wild-type, hel2∆, and upf1∆ cells with (FK)8 DMS library	analysis/barcodeseq/upf1_fk8_dms/scripts/plot_variant_effects_wt_hel2_upf1_reps.ipynb